The disclosed invention is generally in the field of transgenic fish, and more specifically in the area of transgenic fish exhibiting tissue-specific expression of a transgene.
Transgenic technology has become an important tool for the study of gene and promoter function (Hanahan, Science 246:1265-75 (1989); Jaenisch, Science 240:1468-74 (1988)). The ability to express, and study the expression of, genes in whole animals can be facilitated by the use of transgenic animals. Transgenic technology is also a useful tool for cell lineage analysis and for transplantation experiments. Studies on promoter function or lineage analysis generally require the expression of a foreign reporter gene, such as the bacterial gene lacZ. Expression of a reporter gene can allow the identification of tissues harboring a transgene. Typically, transgenic expression has been identified by in situ hybridization or by histochemistry in fixed animals. Unfortunately, the inability to easily detect transgene expression in living animals severely limits the utility of this technology, particularly for lineage analysis.
An attractive paradigm for the understanding of gene expression, development, and genetics of animals, especially humans, is to study less complex organisms, such as Escherichia coli, Drosophila, and Caenorhabditis. The hope is that understanding of these processes in simple organisms will have relevance to similar processes in mammals and humans. The tradeoff is to accept the disadvantage that an experimental organism is only distantly related to humans for the advantage of easy manipulation, fast generation times, and more straightforward interpretation of results in the experimental organism. The disadvantage of this tradeoff can be lessened by using an organism that is as closely related as possible to mammals while retaining as many of the advantages of less complex organisms. The problem is to identify suitable organisms for such studies, and, more importantly, to develop the tools necessary to manipulate such organisms.
Some examples of cell determination in invertebrates have been shown to occur in progressive waves that are regulated by sequential cascades of transcription factors. Much less is known about such processes in vertebrates. An integrated approach combining embryological, genetic and molecular methods, such as that used to study development in Drosophila (for example, Ghysen et al., Genes and Dev 7:723-33 (1993)), would facilitate the identification of the molecular mechanisms involved in specifying neuronal fates in vertebrates, but such an approach has been hampered by a lack of robust genetic and molecular tools for use in vertebrates.
Transgenic technology has been applied to fish for various purposes. For example, transgenic technology has been applied to several commercially important varieties of fish, primarily in an attempt to improve their cultivation. The use of transgenic technology in fish has been reviewed by Moav, Israel J. of Zoology 40:441-466 (1994), Chen et al., Zoological Studies 34:215-234 (1995), and Iyengar et al., Transgenic Res. 5:147-166 (1996).
Stuart et al., Development 103:403-412 (1988), describe integration of foreign DNA into zebrafish, but no expression was observed. Stuart et al., Development 109:577-584 (1990), describe expression of a transgene in zebrafish from SV40 and Rous sarcoma virus transcription regulatory sequences. Although expression was seen in a pattern of tissues, the expression within a given tissue was variegated. Also, since Stuart et al. (1990) selected transgenics by expression and not by the presence of the transgene, non-expressing transgenics would have been missed by their analysis. Culp et al., Proc. Natl. Acad. Sci. USA 88:7953-7957 (1991), describe integration and germ line transmission of DNA in zebrafish. Although the constructs used included the Rous sarcoma virus LTR or SV40 enhancer promoter linked to a lacZ gene, no expression was observed. Bayer and Campos-Ortega, Development 115:421-426 (1992), describe integration and expression in zebrafish of a lacZ transgene having a minimal promoter (a mouse heat shock promoter) but no upstream regulatory sequences. The expression obtained depended on the site of integration indicating that endogenous sequences at the site of integration of the fish were responsible for expression. Westerfield et al., Genes and Development 6:591-598 (1992), describe transient expression in zebrafish of xcex2-galactosidase from mouse and human Hox gene promoters. Lin et al., Dev. Biology 161:77-83 (1994), describe transgenic expression of lacZ in living zebrafish embryos. The transgene linked the enhancer-promoter of the Xenopus elongation factor 1xcex1 gene with the lacZ coding sequence. Different lines of transgenic fish exhibited different patterns of expression, indicating that the site of integration may be affecting the pattern of expression. Amsterdam et al., Dev. Biology 171:123-129 (1995), and Amsterdam et al., Gene 173:99-103 (1996), describe transgenic expression of green fluorescent protein (GFP) in zebrafish. The transgene linked the enhancer-promoter of the Xenopus elongation factor 1xcex1 gene with the GFP coding sequence. As in Lin et al., Dev. Biology 161:77-83 (1994), different lines of transgenic fish exhibited different patterns of expression, indicating that the site of integration may be affecting the pattern of expression. Although some of the systems described above exhibited patterned expression, none resulted in the transmission of stable tissue-specific expression of a transgene in zebrafish.
It is an object of the present invention to provide transgenic fish having tissue- and developmentally-specific expression of transgenes.
It is another object of the present invention to provide a method of making transgenic fish having tissue- and developmentally-specific expression of transgenes.
It is another object of the present invention to provide a method of identifying compounds that affect expression of fish genes of interest.
It is another object of the present invention to provide a method of identifying the pattern of expression of fish genes of interest.
It is another object of the present invention to provide a method of identifying genes that affect expression of fish genes of interest.
It is another object of the present invention to provide a method of genetically marking mutant fish genes.
It is another object of the present invention to provide a method of identifying fish that have inherited a mutant gene.
It is another object of the present invention to provide a method of identifying enhancers and other regulatory sequences in fish.
It is another object of the present invention to provide a construct that exhibits tissue- and developmentally-specific expression in fish.
Disclosed are transgenic fish, and a method of making transgenic fish, which express transgenes in stable and predictable tissue- or developmentally-specific patterns. The transgenic fish contain transgene constructs with homologous expression sequences. Also disclosed are methods of using such transgenic fish. Such expression of transgenes allow the study of developmental processes, the relationship of cell lineages, the assessment of the effect of specific genes and compounds on the development or maintenance of specific tissues or cell lineages, and the maintenance of lines of fish bearing mutant genes.